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Molecular characterization of the fibrinogen-erythrocyte interaction as a cardiovascular risk

Publication . Guedes, Ana Filipa; Santos, Nuno C., 1972-; Carvalho, Filomena A., 1976-

Cardiovascular diseases (CVD) are the major cause of death worldwide. The evaluation of new risk factors can help to identify individuals at higher risk. Erythrocyte aggregation is one of the indicators of cardiovascular risk, which seems to be influenced by high plasma fibrinogen levels. This acute plasma protein has an alternatively spliced γchain variant (γ’ fibrinogen) that has been particularly associated with altered structure or properties of the clots and with increased cardiovascular risk. However, this association has been controversial and the molecular mechanisms are not clear. Based on these findings, we studied how fibrinogen and its variants interact with erythrocytes, and how this binding influences erythrocyte aggregation and fibrin clot properties. After the evaluation and characterization of the fibrinogen-erythrocyte binding in healthy subjects, the main goal of this Thesis was the study of fibrinogen-erythrocyte binding in patients with different cardiovascular and cerebrovascular diseases, as a clinical risk factor, trying to understand the impact of the presence of fibrinogen on erythrocyte aggregation. Clinical and hemorheological profiles, and the changes on γ’ fibrinogen levels associated to CVDs were also evaluated. To reach our goals, different experimental approaches were performed, including atomic force microscopy-based force spectroscopy, magnetic tweezers, ELISA assays, confocal and scanning electron microscopy, hemorheological methodologies and clot permeability studies. Patients with chronic heart failure (CHF), essential arterial hypertension (EAH) and stroke were enrolled in this study. Results were correlated with patient’s clinical profile. Our conclusions are that erythrocytes triggered variability in the fibrin network structure, in the individual fiber characteristics and overall clot properties. The presence of γ’ chain on the fibrinogen molecule, even combined with γA chain, altered the clot structure. Thus, clots with γAγ’ fibrinogen might interact differently with erythrocytes, potentially increasing the risk of thrombosis. Addressing our main goal, we found, at the single-molecule level, that fibrinogenerythrocyte binding forces were higher in EAH, CHF and stroke patients, when compared with the control group, despite of a lower binding frequency. This transient binding is not enough to form a clot, but it could lead to changes on whole blood flow, representing a cardiovascular risk factor. Moreover, a 12-month clinical follow-up showed that CHF patients with higher fibrinogen-erythrocyte binding forces had a significantly higher probability of being hospitalized due to cardiovascular complications in the following year, pointing out the value of this approach for clinical prognosis. Importantly, we also showed that erythrocytes from EAH patients adhere more than those from the control group. Upon increasing fibrinogen concentration, there was an increase in the work and force necessary for cell-cell detachment, both on healthy donors and EAH patients, showing the importance of this plasma protein on the adhesion between these cells. Changes on hemorheological profiles were found in patients compared to the control group. γ’ fibrinogen levels were higher in patients than in healthy donors, which is indicative of an increased cardiovascular risk. Thus, these results are relevant to conclude on the degree of pathophysiological relevance of fibrinogen and erythrocyte aggregation, since an increment on both might induce a state of microcirculatory slower flow, increasing the probability of cardiovascular complications. Moreover, it was concluded that the specific receptor of fibrinogen on erythrocyte membrane is the integrin αvβ3 and the 94RGD97 sequence on the Aα-chain of the fibrinogen is one of the essential binding sites. These results, together with future studies, will be important to discover therapeutic drugs in order to overcome the erythrocyte aggregation promoted by the fibrinogen-erythrocyte binding and, thus, reduce the possibility of cardiovascular events.

2018Doctoral thesis

Open access

The 95RGD97 sequence on the Aα chain of fibrinogen is essential for binding to its erythrocyte receptor

Publication . Carvalho, Filomena Almeida; Guedes, Ana Filipa; Duval, Cedric; Macrae, Fraser L.; Swithenbank, Luke; Farrell, David H.; Ariëns, Robert A. S.; Santos, Nuno C.

BACKGROUND: Erythrocyte aggregation, a cardiovascular risk factor, is increased by high plasma fibrinogen levels. Here, the effect of different fibrinogen mutations on binding to its human erythrocyte receptor was assessed in order to identify the interaction sites. METHODS: Three fibrinogen variants were tested, specifically mutated in their putative integrin recognition sites on the Aα chain (mutants D97E, D574E and D97E/D574E) and compared with wild-type fibrinogen. RESULTS: Atomic force microscopy-based force spectroscopy measurements showed a significant decrease both on the fibrinogen-erythrocyte binding force and on its frequency for fibrinogen with the D97E mutation, indicating that the corresponding arginine-glycine-aspartate sequence (residues 95-97) is involved in this interaction, and supporting that the fibrinogen receptor on erythrocytes has a β3 subunit. Changes in the fibrin clot network structure obtained with the D97E mutant were observed by scanning electron microscopy. CONCLUSION: These findings may lead to innovative perspectives on the development of new therapeutic approaches to overcome the risks of fibrinogen-driven erythrocyte hyperaggregation.

2018Journal article

Open access

Sensing adhesion forces between erythrocytes and γ’ fibrinogen, modulating fibrin clot architecture and function

Publication . Guedes, Ana Filipa; Carvalho, Filomena Almeida; Domingues, Marco; Macrae, Fraser L.; McPherson, Helen R.; Santos, Nuno C.; Ariёns, Robert A.S.

Plasma fibrinogen includes an alternatively spliced γ-chain variant (γ'), which mainly exists as a heterodimer (γAγ') and has been associated with thrombosis. We tested γAγ' fibrinogen-red blood cells (RBCs) interaction using atomic force microscopy-based force spectroscopy, magnetic tweezers, fibrin clot permeability, scanning electron microscopy and laser scanning confocal microscopy. Data reveal higher work necessary for RBC-RBC detachment in the presence of γAγ' rather than γAγA fibrinogen. γAγ' fibrinogen-RBCs interaction is followed by changes in fibrin network structure, which forms an heterogeneous clot structure with areas of denser and highly branched fibrin fibers. The presence of RBCs also increased the stiffness of γAγ' fibrin clots, which are less permeable and more resistant to lysis than γAγA clots. The modifications on clots promoted by RBCs-γAγ' fibrinogen interaction could alter the risk of thrombotic disorders.

2018Journal article

Restricted access

Fibrinogen–erythrocyte binding and hemorheology measurements in the assessment of essential arterial hypertension patients

Publication . Guedes, Ana Filipa; Moreira, Carlos; Nogueira, José B.; Santos, Nuno C.; Carvalho, Filomena Almeida

Some studies have reported a positive association between plasma fibrinogen levels, erythrocyte aggregation and essential arterial hypertension (EAH). The aim of this study was to understand how the interaction between fibrinogen and its erythrocyte membrane receptor is altered in EAH. EAH patients (n = 31) and healthy blood donors (n = 65) were enrolled in the study. EAH patients were therapeutically controlled for the disease, presenting a systolic blood pressure between 108 and 180 mmHg and a diastolic blood pressure between 66 and 123 mmHg. Clinical evaluation included blood pressure monitoring, electrocardiography, echocardiography and blood cell count. The hemorheological parameters were also analyzed. Fibrinogen–erythrocyte binding force and frequency were evaluated quantitatively, at the single-molecule level, using atomic force microscopy (AFM). Changes in erythrocyte elasticity were also evaluated. Force spectroscopy data showed that the average fibrinogen–erythrocyte binding forces increase from 40.4 ± 3.0 pN in healthy donors to 73.8 ± 8.1 pN in patients with EAH, despite a lower binding frequency for patients compared to the control group (7.9 ± 1.6% vs. 27.6 ± 4.2%, respectively). Elasticity studies revealed an increase of erythrocyte stiffness in the patients. The stronger fibrinogen binding to erythrocytes from EAH patients and alteration in cell elasticity may lead to changes in the whole blood flow. The patients’ altered hemorheological parameters may also contribute to these blood flow perturbations. The transient bridging of two erythrocytes, by the simultaneous binding of fibrinogen to both of them, promoting erythrocyte aggregation, could represent an important cardiovascular risk factor.

2019Journal article

Restricted access

Atomic force microscopy as a tool to evaluate the risk of cardiovascular diseases in patients

Publication . Guedes, Ana Filipa; Carvalho, Filomena Almeida; Malho, Maria Inês; Lousada, Nuno; Sargento, Luís; Santos, Nuno C.

The availability of biomarkers to evaluate the risk of cardiovascular diseases is limited. High fibrinogen levels have been identified as a relevant cardiovascular risk factor, but the biological mechanisms remain unclear. Increased aggregation of erythrocytes (red blood cells) has been linked to high plasma fibrinogen concentration. Here, we show, using atomic force microscopy, that the interaction between fibrinogen and erythrocytes is modified in chronic heart failure patients. Ischaemic patients showed increased fibrinogen-erythrocyte binding forces compared with non-ischaemic patients. Cell stiffness in both patient groups was also altered. A 12-month follow-up shows that patients with higher fibrinogen-erythrocyte binding forces initially were subsequently hospitalized more frequently. Our results show that atomic force microscopy can be a promising tool to identify patients with increased risk for cardiovascular diseases.

2016Journal article

Restricted access